AD is at present the most common cause of dementia. It is clinically characterized by a global decline of cognitive function that progresses slowly and leaves end-stage patients bound to bed, incontinent and dependent on custodial care. Death occurs, on average, 9 years after diagnosis (Citron et al., 2004). The incidence rate of AD increases dramatically with age. United Nation population projections estimate that the number of people older than 80 years will approach 370 million by the year 2050. Currently, it is estimated that 50% of people older than age 85 years are afflicted with AD. Therefore, more than 100 million people worldwide will suffer from dementia in 50 years. The vast number of people requiring constant care and other services will severely affect medical, monetary and human resources (Suh & Checker, 2002).
Currently, clinical diagnosis of AD is based on structured interviews (patient histories), and neuropsychological examinations coupled with imaging or neurophysiological scans (CT, MRI, PET and/or SPECT scans and EEG) to rule out other explanations of memory loss including temporary (depression or vitamin B12 deficiency) or permanent conditions (stroke) and is based on NINCDS-ADRDA Work group criteria (McKhann et al., 1984) and the American Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders (4th Ed. Washington D.C., Am Psychiatric Assoc., 1997).
Unfortunately, clinical diagnostic methods are not foolproof. Evidence based review of current literature shows clinical diagnostic accuracy of 65 to 90%. Higher accuracy rates are generally associated with specialized centers (memory disorder clinics) focused on memory disorders whereas lower rates are likely associated with primary care physicians. Additionally, accuracy of the clinical diagnosis is likely lower during early stages of the disease when symptoms are difficult to differentiate from normal age-associated cognitive decline. More recently, studies suggest that a condition termed mild cognitive impairment (MCI) represents prodromal AD and if diagnosed early represents the best opportunity for pharmaceutical intervention. The clinical criteria used for diagnosis of MCI are those of Petersen et al. (1999) and include: 1) memory complaints corroborated by an informant, 2) objective memory impairment for age and education, 3) normal general cognitive function, 4) intact activities of daily living, and 5) the subject does not meet criteria for dementia.
Further complicating diagnosis and treatment of AD is the lack of a reliable biomarker that specifically identifies AD subjects, particularly early in the prodromal stage of the disease (MCI). In view of the magnitude of the public health problem posed by AD, considerable research efforts have been undertaken to elucidate the etiology of AD as well as to identify biomarkers, characteristic proteins or metabolites objectively measured as an indicator of pathogenic processes, that can be used to diagnose and/or predict whether a person is likely to develop AD.
Most studies of biomarkers of AD have focused on measurement in the cerebrospinal fluid (CSF). Because of its intimate contact with the brain, pathogenic changes in the brain that result in alterations in proteins/peptides would likely be reflected in the CSF.
A number of U.S. patents and published applications relate to methods for diagnosing AD, including U.S. Pat. Nos. 4,728,605, 5,874,312, 6,027,896, 6,114,133, 6,130,048, 6,210,895, 6,358,681, 6,451,547, 6,461,831, 6,465,195, 6,475,161, 6,495,335, 2005/0244890, and 2005/0221348. Additionally, a number of reports in the scientific literature relate to certain biochemical markers and their correlation/association with AD, including Fahnestock et al., 2002; Masliah et al., 1995; Power et al., 2001; and Burbach et al., 2004. Additionally, Li et al. (2002) and Sanna et al. (2003) have investigated Leptin in relation to memory and multiple sclerosis, respectively.
Three different biomarkers in CSF have been particularly well documented: neuronal thread protein, tau (total; T-tau and various phosphorylated forms; P-tau) and derivatives of amyloid precursor protein (APP) including Aβ40 and Aβ42 Neuronal thread protein is described to be overexpressed in brain neurons in AD patients. A quantitative test for measuring levels of a specific type of neuronal thread protein (AD7c-NTP) in CSF and urine has been developed. Quite a number of studies have evaluated CSF-tau as an ante-mortem marker for AD mainly using enzyme-linked immunoabsorbent assays (ELISA) as the measurement assay. In past studies, total tau (T-tau) has been measured although there is an increasing body of literature also describing the analysis of phosphorylated (P-tau) variants of the same protein involved in the formation of neurofibrillary tangles. ELISAs that can distinguish between the major form of Aβending at amino acid 40 (Aβ40) and the senile plaque forming species ending at position 42 (Aβ42) have also been developed and evaluated extensively for CSF analysis. These three assays, either used individually, or in the case of tau and Aβ42, in combination, have not demonstrated the required sensitivity and specificity values for routine clinical use, particularly for early diagnosis and discrimination between AD and other non-AD dementias. In addition, attempts to measure tau and Aβ42 in blood have been met with limited success, further restricting their widespread adoption into clinical practice.
A wide spectrum of other aberrations, other than NTP, Tau and Aβ, has been reported in AD patient CSF. Many of the identified (protein sequence confirmed) CSF markers reported herein have been shown to be either increased or decreased in AD patients versus normal individuals. For example, the protein Ubiquitin is known to complex with hyperphosphorylated Tau during maturation of NFTs in the brains of AD patients (Iqbal et al., 1998). Ubiquitin levels in CSF of AD and neurological control groups have been shown to be significantly higher than those of non-neurological aged controls (Wang et. al., 1991; Kudo et al., 1994).
The acute phase/inflammatory protein alpha(1)-antichymotrypsin (ACT) is overproduced in the AD brain. ACT also can promote the formation of, and is associated with, neurotoxic amyloid deposits (Potter et al., 2001). The levels of ACT in both serum and CSF are significantly and specifically higher in patients with Alzheimer-type dementia than in control subjects (Matsubara et al., 1990). There is a particularly close association of increases in CSF-ACT with late onset AD (Harigaya et al., 1995).
Chromogranin A (CrA) is the major protein of large dense-core synaptic vesicles and may be of value as a biochemical marker for synaptic function in AD. One report described no difference between AD, vascular dementia, and age-matched control groups except when comparing a familial subtype (AD Type I) with controls where there was a statistically significant elevation of CSF CrA in the diseased individuals (Blennow et al., 1995).
Beta-2-Microglobulin (β2M) is an initiator of inflammatory responses modulated by interferons and certain cytokines (Hoekman et al., 1985). A proteome analysis of CSF by two-dimensional electrophoresis (2D-gel) has shown a significant increase of β2M in AD patients (Davidsson et al., 2002), and more recently these results were confirmed by SELDI analysis (Carrette et al, 2003).
Transthyretin (TTR) has been shown to interact with A.β, possibly preventing amyloid formation in biological fluids and in the brain. (Tsuzuki et al., 2000). One identified TTR isoform was shown to be increased in AD-CSF using 2D gel analysis of a small number of AD and control patients (Davidsson, supra.). However, this result conflicts with other reports showing a clear decrease of TTR in CSF from AD patients compared with controls (Serot et al., 1997; Riisoen et al., 1998). This decrease is also negatively correlated with the senile plaque (SP) abundance (Merched et al., 1998).
Cystatin C, a cysteine protease inhibitor, has been implicated in the neurodegenerative and repair processes of the nervous system, and the deposition of the same protein together with beta amyloid peptide was found as cerebral amyloid angiopathy (CAA) in different types of dementias (Levy et al., 2001). Full length Cystatin C was found as a CSF marker for AD in a previous SELDI profiling study (Carrette, supra.). A relative blood-brain barrier (BBB) dysfunction is associated with AD among very elderly individuals. The CSF/serum albumin ratio can be used as a measure of BBB function. Mean CSF/serum albumin ratio has been reported to be higher in all dementias studied, including AD, than in nondemented individuals (Skoog et al., 1998).
Transferrin (TF) plays a role in anti-oxidant defense in serum and is also produced in the brain where its role in oxidative stress is unclear. A study on Down's syndrome patients suffering from progressive dementia showed decreased levels of TF when compared to age-matched controls with no neurological disease (Elovaara, 1984).
Prostaglandin-D-Synthase (PDS) functions to convert prostaglandin H2 to prostaglandin D2 and has been identified in several studies of CSF (Harrington et al., 1993; Hiraoka et al., 1998); Hiraoka et al., 2001; Kawashima et al., 2001; Mase et al., 1999; Mase et al., 2003; Melegos et al., 1997. Additionally, PDS demonstrates altered isoforms in neurologic disorders including AD and Parkinson's disease.
Because of the increasing importance of AD in our societies, there is a need for new diagnostic tools and efficient AD biomarkers.